PROJECT SUMMARY The goal of this project is to establish a detailed molecular understanding for how tail-anchored (TA) membrane proteins are post-translationally inserted into the endoplasmic reticulum (ER) membrane. TA proteins, which account for nearly 5% of all eukaryotic membrane proteins, are found in virtually all cell membranes where they play essential roles in diverse cellular processes including intracellular traf?cking, protein translocation, enzyme catalysis and protein quality control. Defects in TA protein biogenesis are linked to many human pathologies, and thus a better understanding of function and dysfunction in these systems may lead to new therapeutic strategies for myriad disease states. Post-translational targeting and insertion of TA proteins into the ER membrane is a multi-step process mediated by the `Guided Entry of Tail-anchored proteins' (GET) pathway, ?rst discovered in early 2007. Since then, my lab has made fundamental contributions towards understanding the molecular basis of TA protein biogenesis in yeast and in mammals. Our rigorous studies performed during the previous granting period recapitulated the early, `pre-targeting' steps of the pathway using completely puri?ed components and established that the essential transmembrane `insertase' (called Get1/2) functions as a heterodimeric complex. In addition, we determined the ?rst high-resolution structures of a functional membrane protein targeting complex; this work resolved what was an ongoing controversy about the nature of the Get3-TA protein complex and de?ned a new paradigm for how transmembrane domains (TMDs) are shielded during transit through the aqueous cytosol. During the course of this project we have assembled a valuable suite of reagents, high-resolution structures, and functional assays that exploit yeast and cell-free systems. Indeed, we have now reconstituted every step in the pathway?from TA protein synthesis to TA protein insertion?using a set of puri?ed, recombinant soluble and membrane components. The power of this system lies in our ability to manipulate each component and step in the pathway, using recombinant and chemical tools. Thus, we are in a unique position to de?ne the structural, biochemical and biophysical principles that underlie every step in the pathway. Here we build on this technical and conceptual foundation to address two central questions that remain poorly understood in the ?eld. In Aim 1, we will de?ne how the Get1/2 transmembrane complex coordinates TA protein insertion into the ER membrane. In Aim 2 we will de?ne how the pre-targeting machinery captures TA proteins and transfers them onto the Get3 targeting factor. We will do this using a multi-disciplinary approach that combines functional analysis with a hybrid computational and experimental structural analysis of soluble and membrane protein complexes.